Volume 18, Issue 11, Pages 1390-1400 (November 2011) Selective and Cell-Active Inhibitors of the USP1/ UAF1 Deubiquitinase Complex Reverse Cisplatin Resistance in Non-small Cell Lung Cancer Cells  Junjun Chen, Thomas S. Dexheimer, Yongxing Ai, Qin Liang, Mark A. Villamil, James Inglese, David J. Maloney, Ajit Jadhav, Anton Simeonov, Zhihao Zhuang  Chemistry & Biology  Volume 18, Issue 11, Pages 1390-1400 (November 2011) DOI: 10.1016/j.chembiol.2011.08.014 Copyright © 2011 Elsevier Ltd Terms and Conditions

Chemistry & Biology 2011 18, 1390-1400DOI: (10. 1016/j. chembiol. 2011 Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 1 qHTS against Human USP1/UAF1 (A) Schematic representation of the USP1/UAF1-catalyzed hydrolysis of ubiquitin-rhodamine110-glycine substrate. (B) Heat maps illustrating the 1536-well plate activity of one representative compound library that was screened from low to high concentrations (left to right) with each plate containing a different compound concentration. The percent activity is depicted as a gradient of color where white, blue, and red indicate no, increasing, and decreasing activity, respectively, relative to no-inhibitor control wells. Calculated Z′-values, the standard statistical parameter for evaluating HTS methods, are indicated below each plate. (C) A three-dimensional scatter plot of the concentration-response curves obtained from the library shown in (B). Percent inhibition was computed from the no-inhibitor (0% inhibited) control and the no-enzyme (100% inhibited) control. Concentration-response relationships are shown for inactive and active compounds in gray and blue, respectively. See also Table S1. Chemistry & Biology 2011 18, 1390-1400DOI: (10.1016/j.chembiol.2011.08.014) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 2 Inhibition of USP1/UAF1 in an Orthogonal Gel-Based Diubiquitin Assay Dose-dependent inhibition of USP1/UAF1 activity (left) and SDS-PAGE analysis of the cleavage of K63-linked diubiquitin (right) in the presence of different concentrations of pimozide (A) and GW7647 (B). See also Figure S1. Chemistry & Biology 2011 18, 1390-1400DOI: (10.1016/j.chembiol.2011.08.014) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 3 Reversibility of Inhibition by USP1/UAF1 Inhibitors (A) SDS-PAGE showing the diubiquitin cleavage by USP1/UAF1 that was preincubated with different inhibitors and then rapidly diluted into assay buffer. (B) The remaining activity of USP1/UAF1 treated with five inhibitors. The bar graph represents mean of three independent experiments ±SD. Pimozide, GW7647, flupenthixol, and trifluoperazine are reversible inhibitors. Rottlerin is largely an irreversible inhibitor. Chemistry & Biology 2011 18, 1390-1400DOI: (10.1016/j.chembiol.2011.08.014) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 4 Inhibition Mechanism of Pimozide and GW7647 Lineweaver-Burk plots of USP1/UAF1-catalyzed hydrolysis of diubiquitin in the presence of 0 (square), 0.5 (circle), 1 (triangle), 2 (diamond) μM of pimozide (A) or GW7647 (B). The inset shows the plot of the slopes obtained from the Lineweaver-Burk plots against the inhibitor concentrations. See also Figure S2. Chemistry & Biology 2011 18, 1390-1400DOI: (10.1016/j.chembiol.2011.08.014) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 5 Cytotoxicity of Cisplatin, Pimozide, and GW7647 Tested as a Single Agent or in Combination on Cisplatin-Resistant NSCLC Cells (H596) (A) Cytotoxicity of cisplatin alone (green diamond), pimozide (blue triangle), or a combination of cisplatin and pimozide at ratios of 1:1 (red circle), 1:4 (black square). (B) Cytotoxicity of cisplatin alone (green diamond), GW7647 (blue triangle), or a combination of cisplatin and GW7647 at ratios of 1:1 (red circle), 1:4 (black square). Cell viability determined with equal volume of vehicle was treated as 100%. Data are mean of three independent experiments ±SD. (C) Combination index (CI) for treating H596 cells with a combination of cisplatin and pimozide or GW7647. Cells were treated with a combination of cisplatin and pimozide at ratios of 1:1 (filled blue triangle), 1:4 (filled blue circle) or with a combination of cisplatin and GW7647 at ratios of 1:1 (open red triangle), 1:4 (open red circle). The combination indices were calculated using software CalcuSyn for each combination and plotted against the fraction of cells affected (Fa). The dashed horizontal line in the figure represents a combination index = 1. See also Figure S3. Chemistry & Biology 2011 18, 1390-1400DOI: (10.1016/j.chembiol.2011.08.014) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 6 Treatment of Human Cells by USP1/UAF1 Inhibitors Leads to the Increase in PCNA and FANCD2 Monoubiquitination HEK293T cells were treated with cisplatin, USP1/UAF1 inhibitors (pimozide or GW7647), or a combination of cisplatin and pimozide or GW7647. The cells treated with the vehicle (DMSO) were used as control. Cell lysate was analyzed by western blotting using anti-PCNA antibody (A) or anti-FANCD2 antibody (B). The PCNA and Ub-PCNA bands were quantified and the percentage of monoubiquitinated PCNA is reported in the bottom panel. The data presented here were an average of three independent experiments. The percentage of monoubiquitinated FANCD2 was determined in the same way. See also Figure S4. Chemistry & Biology 2011 18, 1390-1400DOI: (10.1016/j.chembiol.2011.08.014) Copyright © 2011 Elsevier Ltd Terms and Conditions